Adenovirus serotype 5 (Ad5), which belongs to the C group of human adenoviruses, has been widely used as an oncolytic agent for cancer therapy [1]. Various Ad5 viruses have shown considerable therapeutic effects and have been extensively evaluated in animal models and clinical trials [2]. Their advantage in cancer therapy is due to the self-propagation properties that involve replication in and lysis of infected tumor cells, which leads to secondary infection and killing of adjacent cells within the tumor. A number of therapeutic approaches relying upon adenovirus have been envisioned, including adenoviral vectors expressing therapeutic genes, oncolytic adenoviruses under control of different promoters, which have been described in U.S. Pat. No. 7,951,585, U.S. Pat. No. 7,396,679, U.S. Pat. No. 7,048,920, U.S. Pat. No. 7,078,030 and U.S. Pat. No. 7,001,596, included herein by reference.
One factor limiting the efficacy of Ad5 in cancer therapy is that Ad5 infection is dependent on coxsackievirus-adenovirus receptor (CAR) expression on target cells. CAR is an adhesion molecule expressed in tight-junctions and many cancer cells down-regulate CAR expression, which results in difficulties in achieving sufficient infection and, as a consequence, the oncolytic therapeutic effect is hampered [3]. One approach to circumvent this is to genetically modify Ad5 and use fibers or fiber knobs from the B group of adenoviruses, which do not bind to CAR but to other cell surface receptors [4]. A second limiting factor is fiber-masking of receptors. This is caused by overproduction of adenovirus fiber proteins [5], which are released from the infected cell before cell lysis. The released fibers bind to CAR on non-infected neighboring cells, thereby limiting infection efficiency of progeny virus [5]. The fiber-masking problem is not limited to the Ad5 fiber but has also been observed for the Ad35 fiber, which binds to CD46 [5]. These limitations must be overcome to develop successful oncolytic adenovirus agents.
Cell penetrating peptides (CPPs) are short (usually <30aa) peptides with ability to penetrate tissues or enter cells at a relatively high efficiency. In some cases, members of linear CPPs have the “carrier” features that transport the conjugated “cargos” (from small molecules to large DNA complexes) into cells. Hereafter, CPPs were referred to the class with carrier features unless mentioned specifically. The first insight on cellular uptake of CPPs was discovered in 1965, when researchers reported that histones and basic poly-amino acids stimulate the uptake of albumin by tumor cells in culture. Although the transactivator of transcription (TAT) from HIV-1 virus was the first CPP investigated to determine whether it could function as a carrier, it was not until 1994 that the carrier/penetrating properties of these peptides were fully acknowledged. Further studies by Lebleu's group revealed that the ability to penetrate plasma membranes was associated with certain domains of the TAT protein, which was designated the protein transduction domains (PTD) [6]. Since then an increasing number of new CPPs have been found and characterized. However, the mechanism of uptake is still not fully elucidated, which became the biggest limitation for their transition into clinical applications. Different models have been proposed to explain the penetration into cells. They can be mainly divided into energy-dependent endocytosis and direct translocation via the lipid bilayer. There is also another report suggesting that CPPs only play a role in “adherence” or “docking” to the cell surface while endocytosis mediates the actual cellular uptake. The secondary structure was also found to be important for different classes of CPPs.